Novel Saccharothrix Strain an Antibiotics Derived Therefrom, i.e. Mutactimycins and Aldgamycins

ABSTRACT

The invention relates to a novel strain  Saccharothrix actinomycete  SA 103 deposited at CNCM on 16 Feb. 2004, number 1-3160 or a mutant strain thereof; a method and a medium for selection of said strain; and a method for the production of a broth, active concentrate and active compounds from a culture of said strain SA 103. The invention also relates to active compounds which can be obtained by the production method, i.e. novel mutatimycins and aldgamycins, pharmaceutical compositions comprising said active compounds and the use thereof in medicine and phytopharmaceuticals.

The invention relates to a new actinomycete strain Saccharothrix SA 103registered with the CNCM on 6 Feb. 2004 under number I-3160 or a mutantstrain thereof, a method and a medium for selecting the said strain, amethod for producing a culture medium, an active concentrate and activecompounds from a culture of the said strain SA 103. The invention alsorelates to the active compounds capable of being obtained by theproduction method, particularly novel mutatimycins and aldgamycins,pharmaceutical compositions comprising these active compounds and theiruse in the medical and phytopharmaceutical field.

Antimicrobial agents are widely used in many fields: human health,veterinary medicine, phytopathology, the food industry, leather and woodtreatment, etc. Most of these agents are produced by microorganisms,whereof actinomycetes are among the most important.

Novel antimicrobial agents are always being sought, particularlyantibacterial antibiotics.

In fact, it is known that by the very nature of the bacteria, theyproliferate exponentially, and thereby increase their chances ofbecoming resistant to antibiotics, due to genetic mutations.

The inadequate use of antibiotics in the animal field, particularlyhuman, or plant, and also in the food industry or the leather industry,increases the mutational capacity of the bacteria, and hence theirresistance to antibiotics.

One solution to this problem is obviously to administer the antibioticsjudiciously, but this solution may only be preventive.

A need therefore exists in the present state of the art to develop novelantibiotics. For this purpose, researchers have focused on extremophilicmicroorganisms.

The inventors have thereby isolated a novel actinomycete strainSaccharothrix SA 103 present in the sub-Saharan soil of Algeria, andhave registered it with the Collection Nationale de Cultures deMicroorganisms (CNCM) on 16 Feb. 2004 under number I-3160.

They have also discovered that this strain produced active compounds ofthe class of anthracyclines, some of which have never been previouslyidentified, particularly mutactimycins (PR, F, G), and also activecompounds of the class of anthracylines, particularly aldgamycins (G, Hand P10b). These active compounds have antibacterial activity,particularly against gram positive bacteria, and also antiviral,antiproliferative and anticancer activity.

They therefore developed a method for obtaining these compounds from thestrain Saccharothrix SA 103 registered with the CNCM on 16 Feb. 2004under number I-3160, the said method having the advantage of beingsimple to implement. Thus these antibiotic compounds obtained from thestrain Saccharothrix have a real pharmaceutical interest, both forhumans and for animals, and a plant health interest, and can be producedon the industrial scale.

This is precisely the object of the present invention.

Thus, the primary subject of the invention is the actinomycete strainSaccharothrix SA 103 registered with the CNCM on 16 Feb. 2004 undernumber I-3160 or a mutant strain thereof.

In the context of the present application, “mutant strain”, “mutant”,“variant strain” or “variant”, equally mean a strain of Saccharothrixobtainable by selective mutation from the strain Saccharothrix SA 103 bypreserving the capacity to produce at least one of the active compoundsdescribed below. The mutation techniques are known to a person skilledin the art and consist in placing the strain Saccharothrix SA 103 in thepresence of a physical mutagenic agent, such as radiation, or a chemicalmutagenic agent, for example acriflavin, then selecting in anappropriate medium the remaining mutants of interest by using theirantibiotic spectrum (microorganism inhibition method as, for example,the minimum inhibiting concentration method or MIC, etc.).

The characteristics of the strain Saccharothrix SA 103 according to theinvention are described in the part “Examples” of the presentapplication.

The strain Saccharothrix SA 103 according to the invention or one of itsmutants is cultured in a medium containing a variety of nutrientsubstances generally used for the growth of actinomycetes. For example,as a carbon source, use can be made of glucose, glycerin, sucrose,starch, maltose, or animal or vegetable oils. As a nitrogen source, usecan be made for example of organic nitrogen such as soybean flour, meatextracts, yeast extract, a peptone, maize maceration water, cotton cakeor fish flour. Use can be made of inorganic nitrogen such as, forexample, ammonium sulphate, ammonium chloride, sodium nitrate orammonium phosphate. If necessary, additions can be used of sodiumchloride, potassium chloride, potassium phosphate or divalent metallicsalts such as Mg⁺⁺, Ca⁺⁺, Zn⁺⁺, Fe⁺⁺, Cu⁺⁺, Mn⁺⁺, or Ni⁺⁺, and aminoacids or vitamins. Inclined geloses can be used.

A further subject of the present invention is a method for selecting theactinomycete strain Saccharothrix SA 103 according to the inventionand/or at least one of its mutant strains, characterized in that itcomprises the following steps:

-   -   a) contacting of a biological sample likely to contain the said        strain and/or at least one of its mutant strains with an        appropriate selection medium;    -   b) isolation of the said strain and/or at least one of its        mutant strains.

A further subject of the present invention is an appropriate selectionmedium for isolating the actinomycete strain Saccharothrix SA 103according to the invention and/or at least-one of its mutant strains.

In the present application, appropriate selection medium means anymedium by means of which at least the actinomycete strain SaccharothrixSA 103 according to the invention is capable of developing but whichprevents the growth of at least one strain different from the strainSaccharothrix SA 103 or of another microorganism such as a fungus;preferably, the selection medium according to the invention serves toprevent the growth of all strains different from the strainSaccharothrix SA 103 according to the invention or of a mutant strainthereof.

Such a selection medium comprises a variety of nutrient substances asdefined above and at least one selective product such as, for example,an antibacterial agent to which only the strain Saccharothrix SA 103according to the invention and/or at least one of its mutant strains isresistant, and/or an antifungal agent to eliminate the fungi whereof therapid growth could hinder or even prevent the strain of the inventionfrom growing.

As an example of an appropriate selection medium for isolating thestrain Saccharothrix SA 103 according to the invention, mention can bemade of the M1S medium which corresponds to a humic-vitamin B gelosealso comprising a lysozyme, preferably at a proportion of about 0.005%,cycloserine, preferably at a rate of about 10 mg/l, the pH of thismedium being in the range of about 8.5 to about 9.

Another preferred example of an appropriate selection medium forisolating the strain actinomycete SA 103 according to the invention isthe HV-Vitamin B medium also comprising lysozyme, preferably at the rateof about 0.005%, cycloserine, preferably at the rate of about 10 mg/l,penicillin, preferably at the rate of about 10 mg/l, and rifampicin,preferably at the rate of about 5 mg/l, the pH medium being in the rangeof about 8.5 to about 9.

The selectivity of the appropriate selection medium of the invention canbe reinforced by adding to said medium other selective products to whichthe strain SA 103 according to the invention is resistant, such asparticularly sodium azide, preferably at the rate of about 0.001%,potassium tellurite, preferably at the rate of about 0.01%, and/orcrystal violet, preferably at the rate of about 0.001%.

As an example of an antifungal agent which can be added to theappropriate selection medium of the invention, mention can be made ofactidione and/or nystatin.

When selecting a mutant strain of the strain Saccharothrix SA 103according the to the invention, the selective medium may also,.consistof a culture medium to which a single amino acid has been added, as wellknown to a person skilled in the art.

The strain SA 103 of the invention which the inventors have succeeded inisolating is an extremely rare strain. In fact, a single colony has beenidentified in more than 120 samples analysed and with or withoutselective products such as, for example, the antibiotics mentionedabove.

A further subject of the present invention is a method for producing aculture medium from a culture of the actinomycete strain SaccharothrixSA 103 and/or at least one of its mutant strains according to theinvention, characterized in that it comprises the following steps:

-   -   a) fermentation of the said strain in a nutrient medium to        obtain the culture medium;    -   b) optionally, separation of the culture medium obtained in step        a).

Aerobic fermentation in a liquid medium is preferred, as in the case ofthe production of other antibiotics, and the production of the activecompounds from the strain Saccharothrix SA 103 according to theinvention can be carried out at any temperature favourable to the growthof this strain, that is, ranging from ambient temperature to 43° C.Preferably, use is made of a temperature of between 25° C. and 32° C.Even more preferably, the temperature is 30° C. This culture can lastseveral days, for example from 2 to 10 days. Normally, the pH isslightly alkaline but the exact pH may vary according to the culturemedium used. The strain is taken after growth on gelose, particularlyinclined, and optionally stored at low temperature, and inoculated intoa conventional liquid medium consisting of nutrient substances similarto those described above for the growth of the strain Saccharothrix SA103 according to the invention, with stirring in order to obtain aculture medium.

The fermentation can be carried out in Erlenmeyer flasks and inindustrial or laboratory fermentors having various capacities. Whenfermentation is carried out in a vessel, it is advisable to produce aninoculum in a nutrient culture by inoculating the nutrient culture witha sampling of the culture inclined or flat, or a freeze dried culture ofthe organism. After having obtained an inoculum in this way, it istransferred aseptically to the medium of the fermentation vessel forlarge scale production of the active compounds. The medium in which theinoculum is produced may be the same or may be different from the oneused in the vessel, insofar as appropriate growth of the microorganismis obtained.

The nutrient medium of the invention generally contains the same typesof nutrient substances as those of the culture medium of the strainSaccharothrix SA 103 according to the invention as described above(carbon source, nitrogen source, etc.). It may also contain antifoamingagents such as liquid paraffin, soybean oil, greases or silicone.

The optional step b) for separating the culture medium can be carriedout by any method well known to a person skilled in the art;, alone orcombined with another separation method, such as, for example,centrifugation, filtration or pasteurisation.

Thus preferably, the method for producing a culture medium according tothe invention is characterized in that the separation carried out inoptional step b) is a centrifugation and/or a filtration and/or apasteurization.

A further subject of the invention is a culture medium which can beobtained by the inventive method.

Using the culture medium obtained by the production method of theinvention, the active compounds are extracted. For this purpose, anorganic solvent is used, such as for example, but without limitation,n-butanol, ethyl acetate, dichloromethane, n-propanol or 2-propanol.

After the extraction step, the organic phase can be dehydratedoptionally to remove numerous polar impurities in the activeconcentrate, thereby subsequently facilitating the final purification ofthe active concentrate by HPLC. For this purpose, use can be made ofanhydrous sodium sulphate or magnesium sulphate, and/or drying in vacuoof the organic phase. The use of filtration on gel (cross-linked dextrangel), cellulose column chromatography, an ion exchange resin or thinlayer chromatography (silica gel) can also be considered.

Such methods are well known to a person skilled in the art, who knowshow to use the various techniques alone or in combination, in the mostsuitable way in order to recover an active concentrate from the culturemedium of the strain Saccharothrix SA 103 of the invention.

Thus, a further subject of the present invention is a method forproducing an active concentrate from the culture medium according to theinvention, characterized in that it comprises the following steps:

-   -   a) organic extraction of the culture medium with an organic        solvent;    -   b) optionally, dehydration of the organic phase obtained and/or        drying in vacuo;    -   c) optionally, placing of the active concentrate in suspension,        preferably filtration of the suspension obtained, and repetition        of the steps a) and b) of organic extraction and dehydration.

The active concentrate which can be obtained by the method of thepresent invention is a further subject of the present invention.

The active concentrate thereby obtained is then identified by means ofinstrumental analyses such as visible/ultraviolet absorption spectrum,the infrared absorption spectrum, the ¹H-NMR spectrum and the ¹³C-NMRspectrum, mass spectrometry, and also chromatographic analysis (silicagel or dextran gel chromatographic columns, ion exchange resins, liquidphase chromatography, reverse phase high performance liquidchromatography or HPLC, etc.). This serves to characterise and producethe various active compounds, also called “active fractions” which maybe present in the active concentrate obtained.

Thus a further subject of the invention is a method for producing anactive compound from the active concentrate according to the inventionby reverse phase high performance liquid chromatography (reverse phaseHPLC), preferably preceded by thin layer chromatography and/or lowpressure liquid chromatography.

The active compounds which can be obtained by the production method ofthe invention hence correspond to eluates obtained by HPLCchromatography and are virtually pure.

Purification by HPLC chromatography is indispensable for separating theactive compounds from the active concentrate. Thin layer chromatographyand/or low pressure liquid chromatography such as of the Sephadex LH 20type serves to obtain-an active concentrate stripped of numerousimpurities and thereby facilitates the subsequent purification by HPLCchromatography.

Preferably, the method for producing an active compound according to theinvention is characterized in that the active compound is a mutactimycinsuch as mutactimycin P11, mutactimycin PR, mutactimycin G ormutactimycin F, or an aldgamycin such as aldgamycin G, aldgamycin H oraldgamycin P10b, or the pharmaceutically acceptable addition salts,isomers, enantionmers, diastereoisomers, and mixtures of these activecompounds.

Mutactimycin P11 has the following formula:

It has been identified as correspondent to mutactimycin C, already knownand registered in the “Registry” base under number RN 138689-81-3.

Mutactimycin PR has the following formula:

Mutactimycin F has the following formula:

Mutactimycin G has the following formula:

Aldgamycin G (or P10a) has the following formula:

Aldgamycin with the following stereochemical formula is already known,registered in the “Registry” base under number RN 107745-56-2.

Aldgamycin H (or P8) has the following formula:

Aldgamycin P10b (or swalpamycine B) has the following formula:

A further subject of the invention is an active compound capable ofbeing obtained by the production method according to the invention,characterized in that the active compound is mutactimycin PR having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.

A further subject of the invention is an active compound capable ofbeing obtained by the production method according to the invention,characterized in that the active compound is mutactimycin F having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.

A further subject of the invention is an active compound capable ofbeing obtained by the production method according to the invention,characterized in that the active compound is mutactimycin G having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.

A further subject of the invention is an active compound capable ofbeing obtained by the production method according to the invention,characterized in that the active compound is aldgamycin G having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof,

with the exception of aldgamycin G having the following stereochemicalformula:

A further subject of the invention is an active compound which can beobtained by the production method of the invention, characterized inthat the aldgamycin G has the following stereochemical formula:

or its pharmaceutical acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.

A further subject of the invention is an active compound capable ofbeing obtained by the production method according to the invention,characterized in that the active compound is aldgamycin H having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.

Preferably, the aldgamycin H has the following stereochemical formula:

or its pharmaceutical acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.

A further subject of the invention is an active compound capable ofbeing obtained by the production method according to the invention,characterized in that the active compound is aldgamycin P10b having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.

Preferably, the aldgamycin P10b has the following stereochemicalformula:

or its pharmaceutical acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.

The mutactimycin PR, the mutactimycin F, the mutactimycin G, thealdgamycin G, the aldgamycin H or the aldgamycin P10b according to theinvention can be obtained any method known to a person skilled in theart, such as for example, chemical or enzymatic methods. Preferably, useis made of the production method comprising a fermentation step fromSaccharothrix SA 103 according to the invention as described above.

A further subject of the present invention is an active mixture of atleast two active compounds selected from mutactimycin P11, mutactimycinPR, mutactimycin F, or mutactimycin G, or an aldgamycin such asaldgamycin G, aldgamycin H or aldgamycin P10b.

Optionally, the active mixture according to the invention may contain atleast two active compounds selected among mutactimycin P11, mutactimycinPR, mutactimycin F or mutactimycin G, or an aldgamycin such asaldgamycin G, aldgamycin H or aldgamycin P10b, and another activecompound having antibacterial, antiviral, antiproliferative oranticancer activity known to a person skilled in the art (antibiotics,etc.).

The active compounds of the invention can be used alone or incombination with other microbial agents, for example to prevent thegrowth or reduce the number of gram positive bacteria. Thus thesecompounds are useful from the medical standpoint, particularly for anantibacterial, antiviral or anticancer purpose.

A further subject of the invention is a pharmaceutical compositioncontaining a therapeutically effective quantity of an active compoundaccording to the invention, such as mutactimycin PR, mutactimycin F,mutactimycin G or aldgamycin G, aldgamycin H or aldgamycin P10b and apharmaceutically acceptable excipient.

The pharmaceutical compositions can be prepared in any pharmaceuticallyappropriate form for the administration mode. Examples of suchcompositions comprise solid compositions for oral administration such astablets, capsules, pills, powders and granules, liquid compositions fororal administrations such as solutions, suspensions, syrups andpreparations for parenteral administration such as sterile solutions,suspensions or emulsions.

The active compounds of the present invention form addition salts, ifapplicable pharmaceutically acceptable with reagents for forming suchsalts, well known to a person skilled in the art.

The preferred quantities of the active compounds of the presentinvention used may vary according to the particular active compoundused, the particular composition formulated, the application mode andthe particular site, the host and the disease to be treated. In generalthe active compounds of the invention are injected by theintraperitoneal, intravenous, subcutaneous or local method, oradministered orally. Various factors which modify the action of themedicinal product are taken into account by a person skilled in the art,for example the age, body weight, sex, diet, administration time,excretion rate, condition of the patient, combinations of medicinalproducts, sensitivities to reactions and severity of the disease.Administration can be carried out continuously or periodically in themaximum dose tolerated.

It should be noted that for use as antibacterial agents, the activecompounds are generally administered so that the concentration of theactive ingredient is higher than that of the minimum inhibitingconcentration for the particular organism to be treated.

Thus a subject of the invention is an active compound of the invention,such as mutactimycin PR, mutactimycin F, mutactimycin G or aldgamycin G,aldgamycin H or aldgamycin P10b for its use as a medicinal product.

A further subject of the invention is the use of a pharmaceuticalcomposition according to the invention, for the production of anantibiotic for preventing and/or treating an infection implicating agram positive bacterium, such as a streptococcus, a neonatal infection,a urinary infection, an endocarditis, a pneumonia, a meningitis, anotitis, a listeriosis, diphtheria, tuberculosis or leprosy.

A further subject of the invention is a method for treating and/orpreventing an infection implicating a gram positive bacterium, such as astreptococcus, a neonatal infection, a urinary infection, anendocarditis, a pneumonia, a meningitis, and otitis, a listeriosis,diphtheria, tuberculosis or leprosy, by means of a pharmaceuticalcomposition of the invention.

A further subject is the use of a pharmaceutical composition accordingto the invention, for the production of an antiviral medicinal productfor preventing and/or treating an infection implicating the acquiredimmune deficiency syndrome (AIDS) virus, the vaccine virus, coronavirus, papillomavirus, parvovirus, virus of catarrhal fever of sheep,dengue virus, Ebola virus, or influenza, smallpox, measles, rubella,varicella, hepatitis A, B, C, D or E, mononucleosis, yellow fever,encephalitis or herpes.

A further subject of the invention is a method for treating and/orpreventing an infection of the acquired immune deficiency syndrome(AIDS) virus, the vaccine virus, the corona virus, papillomavirus,parvovirus, virus of catarrhal fever of sheep, dengue virus, Ebolavirus, or influenza, smallpox, measles, rubella, varicella, hepatitis A,B, C, D, or E, mononucleosis, yellow fever, encephalitis or herpes,using a pharmaceutical composition of the invention.

A further subject of the invention is the use of a pharmaceuticalcomposition according to the invention, for the production of ananticancer medicinal product for preventing and/or treating a subjectsuffering from a cancer, such as cancer of the lung, the uterus, breastor ovary, colorectal cancer, leukemia or a subject suffering from atumour of the prostate, the bladder, skin, brain, throat.

A further subject of the invention is the method of treatment and/orprevention of a subject suffering from a cancer, such as cancer of thelung, the uterus, the breast, or the ovary, colorectal cancer, leukemiaor a subject suffering from a tumour of the prostate, the bladder, theskin, brain, throat, using a pharmaceutical composition of theinvention.

A further subject of the invention is a method for preventing as suchand treating a disease in a plant using a phytopharmaceutical productcomprising an active compound according to the invention, such asmutactimycin PR, mutactimycin F, mutactimycin G or aldgamycin G,aldgamycin H or aldgamycin P10b.

The active compounds of the invention such as mutactimycin PR,mutactimycin F, mutactimycin G or aldgamycin G, aldgamycin H oraldgamycin P10b can also be used for treatment in the food, leather orwood industry.

They are also useful in washing solutions for sanitary purposes, forexample for washing hands, and for disinfecting various laboratory,dental and medical equipment or other contaminated materials.

The figure captions and examples below are intended to illustrate theinvention without in any way limiting its scope.

FIGURE CAPTIONS

FIG. 1: Fermentation profile of the strain Saccharothrix sp SA 103.

FIG. 2: Isolation and purification of the active products of the strainSaccharothrix sp SA 103.

EXAMPLES Example 1 Material and Methods

1.1. Microorganism

The productive actinomycete strain Saccharothrix SA 103 according to theinvention was isolated from a soil of the arid regions of Algeria(Sahara) by placing a soil sample in suspension in sterile distilledwater and deposition on a humic-vitamin B gelose containing 50 μg/ml ofactidione. A pure culture of the strain SA 103 was preserved by freezedrying. It was also maintained at 6° C. for use in the laboratory on anISP No. 2 inclined medium (Shirling and Gottlieb, Int. J. Syst.Bacteriol. 16:313-340, 1996).

1.2. Taxonomy

The taxonomic characteristics of the strain SA 103 were determined byculture on various media described in Shirling and Gottlieb (1996) andin Waksman (“The Actinomycetes”, Vol. II, The Williams, & Wilkins Co.Baltimore, 1961). The morphological characteristics were determinedafter growth at 30° C. for 14 days. The colour names and grade numberswere attributed using the ISCC-NBS system (“Inter Society ColourCouncil” for the “National Bureau of Standards”). The detailedobservation of the morphologies of the mycelium and the spores wascarried out by scanning electron microscopy (Hitachi, model S-450). Thephysiological properties were examined using the Goodfellow (J. Gen.Microbiol. 69:33-90, 1971) and Waksman (1961) methods. The type ofisomers of diaminopimelic acid in the cell wall, and the total cellularcomposition of sugars were determined by the methods of Becker et al.(Appl. Microbiol. 12:421-423, 1964), and Lechevalier and Lechevalier(“In the Actinomycetales” Ed. H. Prauser, pp. 311-316, Fisher Verlag,Jena, 1970). Phospholipids and mycolic acids were analyzed by theprocedure of Minnikin et al. (Int. J Syst. Bacteriol. 27:104-107, 1977;J. Chromatography 188:221-233, 1980).

1.3. Fermentation

A sample of the strain SA 103 taken from an immature inclined culturewas inoculated into an Erlenmeyer flask containing 50 ml of the sterileculture medium containing 0.4t glucose, a 1% malt extract and a 0.4%yeast extract (adjusted to pH 7.2 before sterilisation) and placed inculture on a rotary stirrer (250 rpm) at 30° C. for two days. Forproducing antibiotics, 3 ml of culture medium were transferred to 500 mlErlenmeyer flasks, each containing 100 ml of the above medium, andplaced in culture for 10 days using the same conditions. The productionof total antibacterial activity was carried out on a nutrient gelose, bya gelose diffusion test against Bacillus sutilis ATCC 6633. Inhibitionof growth was examined after 24 hours of incubation at 30° C.Antimicrobial activity was estimated by measuring the diameter of theinhibition zone. The dry weight of the mycelium was determined inEppendorf tubes filled with 1 ml of homogenized culture medium and driedat 105° C. for 24 hours (Pfefferle et al. J. Biotech. 80:135-142, 2000).

1.4. Purification of Antibiotics

The production of antibacterial antibiotics by the strain SA 103 wascarried out in liquid medium MS+starch (1%)+yeast extract (0.36) at pH7.2 (100 ml of medium per 500 ml Erlenmeyer flask, stirring at 250 rpm,incubation at 30° C.). A kinetics showed that the strain SA 103 beginsto produce antibiotics from the onset of the exponential phase (1^(st)day) and this production continues to the onset of the steady statephase before stabilising and then decreasing during the decline phase(cf. FIG. 1).

On the whole, eight litres of culture were prepared. The filtrates wereextracted with n-butanol. The butanolic extract containing the desiredantibacterial antibiotics was retained.

The butanolic extract, of a red colour, was concentrated on theRotavapor. An aliquot of this extract was first sent through a SephadexLH 20 column using methanol in double distilled water (80%) as themobile phase. However, the separation of the antibiotics (visualisationby the naked eye and by the antibiography) was not conclusive. This stepwas therefore eliminated and the butanolic extract was purified directlyby a reverse phase HPLC using a C18 column and isocratic conditions (63%methanol in water), a flow rate of 1.5 ml/min and detection at 220 nm.The fractions corresponding to all the peaks obtained on the profilewere collected separately, concentrated and tested against Bacillussubtilis. Seven fractions (7 peaks) proved to be active: four of abright red colour (P11, PR, F and G) and three colourless (P8, P10a andP10b). These antibiotics were purified by HPLC after 3 to 4 reinjectionsin the same conditions as previously.

Example 2 Results and Discussions—Taxonomic Characterisation of theProductive Strain

2.1. Morphology of the Strain

The strain SA 103 formed a well developed pink aerobic mycelium whichbroke into chains of straight or flexible spores. These spores were rodshaped and had a size of 1.9-2.9×0.6-0.7 microns with a smooth surface(FIG. 1). The endospores, scleral granules, synnemata and flagellatespores were not observed. The substrate mycelium was brownish red todeep red and showed few or no fragments. The strain produced acharacteristic abundant dark red pigment which was revealed ascorresponding to antibacterial antibiotics.

2.2. Structural Characteristics

Table 1 shows the culture characteristics of the strain SA 103 onvarious culture supports. The growth of the strain was abundant on yeastextract and malt extract gelose, Bennett gelose and nutrient gelose, butwas moderate on oatmeal gelose and inorganic salts-starch gelose. Thecolour of the mycelium fit into the range between yellowish pink tolight reddish brown for the aerobic hyphae and brownish orange to verydark red for the substrate mycelium. The strain produced a dark red orbrownish orange soluble pigment on all the mediums used but no melanoidpigment was observed.

The least different species was Saccharothrix syringae which has awhitish-pinkish aerobic mycelium, a substrate mycelium andviolet-red-brown soluble pigments.

2.3. Chemotaxonomy

The chemotaxonomic study showed the presence of meso-diaminopimelic acidand the absence of glycine (type III cell wall). The sugar motif of theoverall cell consisted of rhamnose and galactose (type E cell sugar)(Kroppendstedt, “The genus Nocardiopsis. In the Procaryotes” Ed., A.Balows et al., pp. 1139-1159, Springer Verlag, Berlin, 1192)and thecharacteristic phospholipid was phosphatidyl-ethanolamine (phospholipidstype PII). No mycolic acid was detected.

2.4. Physiology of the Strain

The results of the physiological tests are given Table 2.

The strain is capable of using several organic compounds (casein,gelatine, tween 80, starch, tyrosine, etc.) including most sugars. Incontrast, with the exception of mannitol, the alcohol derivatives of theoses (inositol, adonitol, dulcitol, erythritol, and sorbitol) are notdegraded. The strain is capable of growing at 20° C. and 48° C. (with anoptimum at 30° C.) and at pH 5 and 9 (with an optimum between 7 and 8).It is resistant to the lysozyme and to crystal violet but is sensitiveto all the antibiotics tested (11) with the exception of penicillin andrifampicin.

The strain SA 103 is distinguished from Sa. Syringae by its capacity todegrade lactose but not hypoxanthine and sodium butyrate, itssensitivity to erythromycin, gentamicin, oxytetracyclin, and vancomycin,its resistance to penicillin, rifampicin and crystal violet, and itsgrowth at pH 5.

Accordingly, the strain SA 103 could be a novel species of Saccharothrixor possibly a novel subspecies of Sa. Syringae.

2.5. Classification

Based on the morphological and chemical properties described above, itwas considered that the strain SA 103 belonged to the genusSaccharothrix (Labeda, et al, Int. J. Syst. Bacteriol. 34 :426-431,1984). Compared to the nearest species Saccharothrix synrigae NRRL B-16₄₆₈ ^(T), the strain SA 103 differed in its capacity to degrade lactosebut not hypoxanthine and sodium butyrate, its susceptibility toerythromycin, gentamycin, oxytetracyclin and vancomycin, its resistanceto penicillin, rifampicin, crystal violet and sodium azotide and thegrowth of pH 5.0. The strain was accordingly designated Saccharothrixsp. SA 103.

2.6. Fermentation

The variation over time of the production of antibacterial activity bySaccharothrix sp. SA 103 is shown in FIG. 1. The production ofbiological activity against B. subtilis began during the first day andreached its peak on day 4 and then became stable. The biomass increasedduring the first three days and remained stable, then decreased afterday 8. The pH kinetics showed a striking increase during the first day,then became neutral, and rose at the end of fermentation. In general,the production of secondary metabolites by the microorganisms occursduring the steady state phase but in the present case, the production ofbiological activity was strongly correlated to growth and was observedthroughout the variation over time. The same kinetics of production overtime was observed for the production of dithiolopyrolone antibiotics bySaccharothrix sp. SA 233 (Lamari et al., J. Antibiotics 55:696-701,2002) and the production of clavulanic acid by Streptomyces clavuligerus(Lebrihi et al., Appl. Microbiol. Biotechnol. 26:-130-135, 1987).

2.7. Determination of the Structure of the Antibiotics

The various steps in isolation and purification of antibiotics aresummarised in FIG. 2.

2.7.1. Antibiotics of the Anthracyclin Family

The antibiotics P11, PR, F and G are bright red in colour at neutral pH,yellow at acid pH and violet-blue at basic pH. P11 is the majorityantibiotic and PR, the minority antibiotic. These antibiotics were thesubject of the following rectoscopic analyses: UV visible light,infrared, mass spectrometry and NMR of the proton and carbon 13 (withstudies of the correlations). Supplementary tests, such a solubility,determination of Rf by CCM and elemental analysis (the latter only forP11) were also carried out.

Identification of the P11 Molecule with Mutactimycin C

The P11 molecule has a molecular weight of 530 and the chemical formulaC₂₇H₃₀O₁₁. The maxima obtained in the UV-visible (MeOH) are: 219, 234,250, 287, 478, 496 and 531 mm. The spectrum is similar to that of theantibiotics of anthracycline family, and also the colour changes atdifferent pH. The infrared spectrum suggests the presence of aromatics,hydroxyl, methyl and methoxyl groups. Moreover, the final structure waselucidated after a detailed study by NMR of the proton and of carbon 13(chemical shifts, spin-spin coupling constants, signal intensities and¹H—¹H cosy 45, ¹H—¹³C HMCQ and ¹H—¹³C HMBC correlations). The P11molecule was thus identified as mutactimycin C, known to be secreted bya mutant of Streptomyces sp. Mutactimycin C consists of the anthracyclinnucleus (4 adjoining rings including a quinonic ring between two benzenerings), linked (via the carbon C7) to an ose(6-deoxy-3-O-methyl-a-mannopyranoside). P11 is highly soluble in water,methanol, n-butanol, ethanol, 1-propanol and acetone and insoluble inn-hexane and toluene.

Determination of the Chemical Structure of Novel Mutactimycins

Mutactimycin PR

The minor PR molecule has a molecular weight of 662 which corresponds tothe formula C₃₂H₃₈O₁₅. The maxima obtained in the UV-visible and theinfrared bands are identical to those of P11 and the mass fragments arefairly similar between the two antibiotics, suggesting a strongresemblance in the chemical structure. PR is highly soluble in water,methanol and ethanol and insoluble in chloroform, dichloromethane, ethylacetate, n-hexane and toluene. The NMR of the proton and of carbon 13served to elucidate the structure of PR which proved to be close to P11(mutactimycin C) with the only difference being the presence of a secondose linked to the carbon for the 1^(st) benzene ring (in the place of—OCH₃). The second ose is 6-deoxy-mannopyranoside (difference from thefirst by the absence of a methyl) Due to the structure, the PR moleculebelongs to the anthracyclin family and to the group of mutactimycins.However, it differs from all the known mutactimycins and hencerepresents a novel antibiotic of this group, called mutactimycin PR.

Mutactimycin F

The antibiotic F has a molecular weight of 516 (thus 14 less than P11)corresponding to a molecular formula C₂₆H₂₈O₁₁. Its UV-visible andinfrared spectra and its mass fragments are very similar to those of P11and PR. Hence, this suggests a strong resemblance between the threemolecules. The F compound is soluble in water, n-butanol, 1-propanol andethanol and insoluble in n-hexane and toluene. The NMR of the proton andthe carbon 13 served to establish the structure of the molecule whichproved to be very similar to P11 (mutactimycin C), with the soledifference of the presence of an OH on the carbon C3 of themannopyranosyl residue, instead of a —OCH3 group. The F antibiotic isdifferent from all known mutactimycins and represents a novel moleculecalled mutactimycin F.

Mutactimycin G

The molecule has a molecular weight of 502 corresponding to a formulaC₂₅H₂₆O₁₁. Strong similarities between G, F, PR and P11 are observed inthe UV-visible and infrared spectra, and the mass fragments. The Gproduct is soluble in water, n-butanol, 1-propanol, methanol and ethanoland insoluble in chloroform, dichloromethane, ethyl acetate, n-hexaneand toluene. The NMR of the proton and carbon 13 served to elucidate thestructure of this molecule, which proved to be very similar to PR, withthe only difference being the absence of a second ose linked to thecarbon C7 of the 4^(th) ring of the anthracycline nucleus. The Gantibiotic hence represents a novel molecule of the mutactimycin groupand was therefore called mutactimycin G.

2.7.2. Antibiotics of the Macrolide Family

The antibiotics P8, P10a and P10b are not coloured. P8 is the majoritymolecule produced by Saccharothrix sp. SA 103 after P11 (mutactimycinC). The three antibiotics are highly soluble in methanol, n-butanol,ethanol, 1 and 2-propnol, ethyl acetate, acetone, chloroform anddichloromethane, but insoluble in water and n-hexane. These antibioticswere the subject of the same spectroscopic analyses as the mutactimycinsdescribed earlier, which served to culminate in the determination oftheir chemical structures.

Identification of P10a with Aldgamycin G

The P10a antibiotic has a molecular weight of 740 corresponding to themolecular formula C₃₇H₅₆O₁₅. The UV-visible spectrum is very similar tothat of aldgamycins and showed a peak at 216 and two shoulders 245 and278 nm. The infrared spectrum shows several absorption bands indicatingthe presence of methyl, methoxyl, hydroxyl groups and a carbonatefunction. P10a is soluble in n-butanol, 1 and 2-propanol, methanol,dichloromethane, ethyl acetate, chloroform, acetone and ethanol, andinsoluble in n-hexane, toluene and water. The P10a molecule was thusidentified with aldgamycin G, known to be secreted by Streptomycesavidinii. Aldgamycin G is a neutral macrolide whereof the lactonic ringcomprises 16 atoms to which are connected two methylated sugars, mycinoein position C14 and aldgarose in position C5.

Determination of the Chemical Structures of the Novel Macrolides

Aldgamycin H

The P8 antibiotic has a molecular weight of 714, thus a molecularformula C₃₆H₅₈O₁₄. Its physicochemical properties are very similar tothose of P10a (aldgamycin G). However, the UV-visible spectrum of P10bshows a lower absorbance intensity at 245 and 278 nm and its infraredspectrum no longer contains the absorption band at 1800 cm⁻¹characteristic of the carbonate group as in the case of aldgamycin G.The product P8, after analysis of all its spectroscopic data, wasidentified with a new aldgamycin called H which is similar to aldgamycinE, but differs in the absence of the carbonate function on the aldgarosesugar which is hydrolyzed in this case.

Swalpamycin B

The P10b molecule could not be separated from the P10a product by HPLCand accordingly, it was analysed at the same time as the product P10a inthe form of a complex. It has a molecular weight of 698 and the chemicalformula C₃₆H₅₈O₁₃. The NMR analyses of the proton and carbon 13 servedto elucidate the final structure. Thus P10a has a chemical structurevery similar to that of P8 and of swalpamycin.

Microbiological Activities of the Antibiotics

The microbiological activity of the mutactimycins P11, PR, F and G andthe macrolides P8, P10 (a and b) is mainly directed against the grampositive bacteria. The most sensitive bacteria are Micrococcus luteusand Klebsiella pneumoniae which is the only gram negative bacteria to besensitive.

The minimum inhibiting concentrations (MIC) are 5 μg/ml for M. luteusand K. pneumoniae, 10 pg/ml for Staphylococcus aureus CIP 53156, 40μg/ml for Bacillus subtilis, 50 μg/ml for Listeria monocytogenes and 75μl/ml for Mycobacterium smegmatis. The novel molecules PR, F and G areactive against the same microorganisms (with the exception of L.monocytogenes).

None of the molecules have any action against S. aureus CIP 7625, thegram negative bacteria Escherichia coli, Pseudomonas syringae pathovarsyringae and Agrobacterium tumefasciens and against the yeastSaccharomyces cerevisiae and the filamentous fungus Mucor romannianus.

The activity of the macrolides (aldgamycins G and H) and swalpamycin Bis greater than those of the mutactimycins. In fact, the MIC are only0.1 μg/ml for K. pneumoniae, 1 μg/ml for Bacillus subtilis, Micrococcusluteus and Staphylococcus aureus CIP 53156 which are the most sensitiveand respectively 30 and 50 μg/ml for Listeria monocytogenes andMycobacterium smegmatis. The gram negative bacteria (except K.pneumoniae) and the fungi are resistant. MIC (μg/ml) Test organisms(swalpamycin) Bacillus subtilis ATCC 6633 1 Micrococcus luteus ATCC 93141 Staphylococcus aureus CIP 7625 >100 Staphylococcus aureus CIP 53156 1Listeria monocytogenes CIP 82110 30 Mycobacterium smegmatis ATCC 607 50Klebsiella pneumoniae CIP 82.91 0.1 Escherichia coli ATCC 10536 >100Pseudomonas syringae No 1882 >100 Agrobacterium tumefasciens No2410 >100 Mucor ramannianus NRRL 1829 >100 Saccharomyces cerevisiae ATCC4226 >100

MIC (μg/ml) of Test organisms (aldgamycin H) Bacillus subtilis ATCC 663310 Micrococcus luteus ATCC 9314 1 Staphylococcus aureus CIP 7625 >100Staphylococcus aureus CIP 53156 5 Listeria monocytogenes CIP 82110 >100Mycobacterium smegmatis ATCC 607 20 Klebsiella pneumoniae CIP 82.91 0.5Escherichia coli ATCC 10536 >100 Pseudomonas syringae No 1882 >100Agrobacterium tumeffasciens No 2410 >100 Mucor ramannianus NRRL1829 >100 Saccharomyces cerevisiae ATCC 4226 >100

TABLE 1 Cultural Characteristics of Saccharothrix sp. SA 103. AerobicSubstrate Diffusible Medium Growth Mycelium Mycelium Pigment Yeast GoodAbundant Very dark Blackish extract-agar moderate red (14) red (16) maltextract yellowish (ISP No. 2) pink (29) Agar oatmeal Moderate ModerateBrownish Brownish flour (ISP pale orange orange No. 3) yellowish (54)(54) pink (31) Inorganic Moderate Moderate Brownish Brownish salts-agarbrownish orange orange starch (ISP pink (33) (54) (54) No. 4) Bennettagar Good Abundant Dark Dark pale reddish reddish reddish brown (44)brown (44) brown (42) Nutrient Good Moderate Dark red Dark red gelose to(13) (13) abundant yellowish pink (29)

TABLE 2 Physiological Properties of the Strain Saccharothrix sp. SA 103.Degradation of − Adenin Arbutin + Casein + Gelatin + Esculin + Guanine −Hypoxanthin − Starch + Testosterone + Tween 80 + Tyrosin + Xanthin −Adonitol − L-Arabinose + Cellobiose + Dextrin + Dulcitol − Erythritol −D-Fructose + Galactose + D-Glucose + Glycerol + Inositol − Lactose −Maltose + D-Mannitol + D-Mannose + Melezitose − Melibiose −α-Methyl-D-glucoside − D-Raffinose − L-Rhamnose + Ribose + Sorbitol −Saccharose + Trehalose + D-Xylose + Nitrate reduction + Production ofmelanoid − pigments Sodium decarboxylation: + Acetate − Benzoate −Butyrate + Citrate − Oxalate + Propionate + Pyruvate + Succinate −Tartrate Growth at: + 48° C. + pH 5 + pH 9 Tolerance to: + Crystalviolet (0.001%) + Lysozyme (0.005%) + Phenol (0.05%) − Phenol (0.1%) +Potassium tellurite + (0.01%) Sodium azide (0.001%) − Sodium azide(0.01%) − Sodium chloride (5) Resistance to: − Chloramphenicol (25μg/ml) − Cycloserin (10 μg/ml) − Erythromycin (10 μg/ml) − Gentamicin(10 μg/ml) − Kanamycin (25 μg/ml) − Novobiocin (10 μg/ml) +Oxytetracyclin (25 μg/ml) + Penicillin (25 μg/ml) − Rifampicin (5 μg/ml)− Streptomycin (10 μg/ml) − Vancomycin (5 μg/ml)

TABLE 3 Antimicrobial Spectrum of Antibiotics PR and P11: MIC (μg/ml) PRP11 Test strains (1) (2) Bacillus subitlis ATCC 6633 75 40 Micrococcusluteus ATCC 9314 50 5 Staphylococus aureus CIP 7625 >100 >100Staphylococcus aureus CIP 53156 50 10 Listeria monocytogenes CIP82110 >100 50 Mycobacterium smegmatis ATCC 607 >100 75 Klebsiellapneumoniae CIP 82.91 40 5 Escherichia coli ATCC 10536 >100 >100Pseudomonas syringae No 1882 >100 >100 Agrobacterium tumefaciens No2410 >100 >100 Mucor ramannianus NRRL 1829 >100 >100 Saccharomycescerevisiae ATCC 4226 >100 >100

TABLE 4 Physicochemical Properties of the Products PR and P11(mutactimycin) mutactimycin PR P11 (mutactimycin C) Appearance Redpowder Red powder Colour in H₂O Acid Yellow Yellow Neutral Red Red BasicViolet-blue Violet-blue Chemical formula C₃₂H₃₈O₁₅ C₂₇H₃₀O₁₁ Molecularweight 662 530 Nano-ESI-MS (m/z) Negative 660.8 [M − H]⁻, 528.8 [M −H]⁻, 351, mode 514.9, 354, 337, 333, 315, 294.2, 319.1, 291.1 293.1Positive 685.2 [M + Na]⁺ 539, 553.1 [M + Na]⁺, 375, mode 507, 360.9,342.9 356.9, 198.3 Uvλ_(max)nm in MeOH 219, 234, 250, 219, 234, 250,287, 287, 478, 496, 531 478, 496, 531 IRν_(max) time in cell 3393, 2969,2932, 3393, 2969, 2932, (cm⁻¹) 2878, 2841, 2709, 2878, 2841, 2709, 2360,2113, 1611, 2360, 2113, 1611, 1583, 1444, 1408, 1583, 1444, 1408, 1379,1352, 1285, 1379, 1352, 1285, 1238, 1213, 1133, 1238, 1213, 1133, 1110,1070, 1048 1110, 1070, 1048 Relative solubility Highly MeOH, EtOH, H₂OMeOH, Me₂CO, H₂0, n- soluble BuOH, EtOH, 1-PrOH Medium Me₂CO, n-BuOH, 1-CH₂Cl₂, CHCl₃, EtOAc, soluble PrOH, 2-PrOH 2-PrOH Insoluble CH₂Cl₂,CHCl₃, n-hexane, toluene EtOAc, n-hexane, toluene CCM (Value of Rf)^(a)(I) 0.16 0.44 (II) 0.58 0.64 (III) 0.82 0.80 HPLC (Rt)^(b) 31.74 min35.65 min^(a)CCM on silica gel (Merek No 5715. (I): EtOAc—MeOH (100:15). (II):n-BuOH—CH₃COOH—H₂O (3:1:1). (III): MeOH—CH₂Cl₂ (4:1).^(b)HPLC conditions: Uptishpere C₁₈ UP5ODB (250 × 7.8 mm, i.d.), Mobilephase: isocratic at 63% MeOH in H₂O, Flow rate: 1.5 ml/min, Detection:UV (220 mm).

TABLE 5 Assignments of NMR data of ¹H and ¹³C of the Product PR inDMSO-d6 at 298 K and in DMF-d₇ at 278 K. DMSO DMF Position δH δC δH δC 1 8.00(d) 121.0  7.96(d) 120.6  2 7.84(br dd) nd 7.81(dd) 135.3  37.66(br) nd 7.68(d) 124.1  4 nd 157.9  4a nd 122.6  7 4.89(m) 73.24.92(m) 73.7  8 2.10(dd)/ 43.3 2.16(dd)/ 43.6 1.92(dd) 1.98(dd)  9 67.867.7 10 2.83(d)/2.61(d) 38.3 2.84(d)/ 38.2 2.65(d) 12a nd 136.0 6-OH ndnd 9-OH 4.70(br) nd 9-Me 1.30(s) 29.7 1.32(s) 29.2 11-OH nd nd  1′5.16(br s) 104.4  5.22(br 104.7 s)  2′ 3.83(m) 67.0 3.92(m) 67.1  3′3.00(dd) 81.5 3.04(dd) 81.7  4′ 3.33^(a) 71.0 3.44(dd) 72.0  5′ 3.64(do)70.2 3.70^(a) 69.8 2′-OH 4.88 nd 3′-OMe 3.22(s) 56.9 3.17(s) 56.6 4′-OH4.90 nd 5′-Me 1.21(d) 18.7 1.17(d) 18.0  1″ 5.69(br s) 99.1 5.73(br 99.4s)  2″ 4.00(m) 71.0 4.09(m) 71.3  3″ 4.04(dd) 70.9 4.10(dd) 71.3  4″3.35^(a) 72.4 3.42(dd) 72.2  5″ 3.52(dq) 70.9 3.54^(a) 70.8 2″-OH5.15(br) nd 3″-OH 4.89(br) nd 4″-OH 4.97 nd 5″-Me 1.09(d) 18.7 1.05(d)17.9The detector signals are represented.^(a)Signal under residual HOD, nd: not detected.

TABLE 6 Assignments of NMR data of ¹H and ¹³C of product P11 in DMSO-d6at 298 K. Position δH δC  1 7.81(m) 119.4  2 7.83(m) 136.0  3 7.56(m)119.1  4 161.5  4a 120.8  5 186.9  5a 111.1  6 157.4  6a 136.0  74.86(dd) 73.5  8 2.13(dd)/ 42.8 1.93(dd)  9 68.2 10 2.76(d)/ 37.42.62(d) 10a 136.8 11 155.4 11a 111.4 12 187.4 12a 135.7 4-ONe 3.95(s)56.2 6-OH 14.13(s)^(a) 9-OH 4.78(s) 9-Me 1.31(s) 28.1 11-OH 13.19(s)^(a) 1′ 5.12(d) 104.2  2′ 3.87(m) 67.1  3′ 3.02(dd) 81.2  4′ 3.32(ddd) 71.7 5′ 3.63(dq) 69.8 2′-OH 4.82(d) 3′-OMe 3.23(s) 56.1 4′-OH 4.91(d) 5′-Me1.21(d) 17.0^(a)Signals which can be interchangeable

TABLE 7 Physicochemical Properties of the Compound F. Mutactimycin FAppearance Bright red powder Chemical formula C₂₆H₂₈O₁₁ Molecular weight516 Nano-ESI-MS (m/z) Negative mode 514.9 [M − H]⁻ Positive mode 539.11[M + Na]⁺ UVλ_(max) nm in MeOH 219, 234, 252, 286, 473, 494, 531IRγ_(max) diamond cell (cm⁻¹) 3351, 2970, 2927, 2854, 2360, 2336, 2114,1981, 1798, 1610, 1582, 1444, 1406, 1381, 1356, 1271, 1240, 1215, 1137,1091, 1069, 1048 Relative solubility Highly soluble n-BuOH, 1-PrOHMedium soluble MeOH, EtOH Insoluble H₂O, CH₂Cl₂, CHCl₃ EtOAc, Me₂CO,n-hexane, toluene CCM (value of Rf)^(a) (I) 0.30 (II) 0.62 (III) 0.78HPLC (Rt)^(b) 22.25 min^(a)CCM on silica gel (Merch No 5715). (I): EtOAc—MeOH (100:15). (II):n-BuOH—CH₃COOH—H₂O (3:1:1). (III): MeOH—CH₂Cl₂ (4:1).^(b)HPLC conditions: Uptisphere C₁₈ UP5ODB (250 × 7.8 mm, i.d.), Mobilephase: isocratic at 63% MeOH in H₂O, Flow rate: 1.5 ml/min, Detection:UV (220 nm).

TABLE 8 Assignments of NMR Data of ¹H and ¹³C of Product F in DMSO-d6 at298 K Position δH δC  1 7.91(m) 120.5  2 7.90(m) 137.0  3 7.64(m) 119.8 4 161.6  4a 119.8  5 187.4  5a 111.4  6 157.5  6a 137.3  7 4.90(dd)73.2  8 2.13(dd)/ 43.3 1.94(dd)  9 67.8 10 2.83(d)/ 38.3 2.64(d) 10a137.0 11 155.5 11a 111.5 12 187.3 12a 135.7 OMe-4 3.99(s) 57.5 OH-614.16(br s)^(a) OH-9 4.78(s) Me-9 1.30(s) 29.8 OH-11 13.26(br s)^(a)  1′5.07(d) 104.6  2′ 3.61(m) 71.4  3′ 3.30(ddd) 71.5  4′ 3.24(ddd) 72.7  5′3.60(dq) 70.2 OH-2′ 4.78(d) OH-3′ 4.47(d) OH-4′ 4.77(d) Me-5′ 1.20(d)19.8^(a)Signal under residual HOD.nd: not detected

TABLE 9 Physicochemical Properties of Compound G. G Appearance Brightred powder Chemical formula C₂₅H₂₆O₁₁ Molecular weight 502 Nano-eSI-MS(m/s) Negative mode 500.9 [M − H]⁻, 355, 337, 319.2, 291.2 UVλ_(max) nmin MeOH 219, 234, 252, 286, 473, 494, 531 Irγ_(max) diamond cell 3351,2970, 2927, 2854, 2360, 2336, 2114, 1981, 1798, 1610, 1582, 1444, 1406,1381, 1356, 1271, 1240, 1215, 1137, 1091, 1069, 1048 Relative solubilityHighly soluble n-BuOH, l-PrOH Medium soluble MeOH, EtOH Insoluble H₂O,CH₂Cl₂, CHCl₃, EtOAc, Me₂CO, n-hexane, toluene CCM (value of Rf)^(a) (I)0.30 (II) 0.62 (III) 0.78 HPLC (Rt)^(b) 22.24 min^(a)CCM on silica gel (Merch No 5715). (I): EtOAc—MeOH (100:15). (II):n-BuOH—CH₃COOH—H₂O (3:1:1). (III): MeOH—CH₂Cl₂ (4:1).^(b)HPLC conditions: Uptisphere C₁₈ UP5ODB (250 × 7.8 mm, i.d.), Mobilephase: isocratic at 63% MeOH in H₂O, Flow rate: 1.5 ml/min, Detection:UV (220 nm).

TABLE 10 Assignments of NMR Data of ¹H and ¹³C of Product G in DMSO-d6at 298 K Position δH δC  1 7.97(dd) 120.9  2 7.87(dd) 137.0  3 7.72(dd)124.2  4 157.8  4a 121.2  5 nd  5a 111.1  6 nd  6a 139.6  7 5.08(dd)63.7  8 2.12(dd)/ 44.3 1.74(dd)  9 68.1 10 2.73(br 38.3 s) 10a 136.6 11155.4 11a 111.4 12 186.2 12a 135.5 OH-6 14.20(br s)^(a) OH-7 4.89(s)OH-9 4.60(s) Me-9 1.31(s) 30.7 OH-11 13.30(br s)^(a)  1′ 5.65(br 99.5 s) 2′ 4.02(m) 70.6  3′ 4.02(m) 71.0  4′ 3.36(m) 72.5  5′ 3.55(dq) 70.9OH-2′ 5.15(d) OH-3′ 4.86(d) OH-4′ 4.96(d) Me-5′ 1.10(d) 19.8^(a)Signal under residual HOD.nd: not detected

TABLE 11 Assignments of NMR Data ¹H and ¹³c of the Product P8(aldgamycinH) in CD₃OD and in DMSO-D₈ at 298 K P8(aldgamycin P8(aldgamycin H) H) inDMSO in CD3OD Position dH(multi, J in Hz) dH(multi, J in Hz) dC  1 166.4 2 6.09 d(15.3) 6.03 d(15.4) 120.9  3 6.56 dd(15.3; 10.6) 6.64 dd(15.4;10.6) 152.1  4 2.72 ddq(10.6; 2.83 ddq(10.6; 41.9 10.2; 6.8) 10.2; 6.7) 5 3.27 br d(10.2) 3.38 br d(10.2) 86.8  6 0.98 br m 1.14 br m 34.6  71.40 ddd(14.4, 1.59 ddd(14.0, 32.0 12.0, 4.5)/1.77 12.0, 4.1)/1.81ddd(14.4, 11.0, ddd 14.0, 11.6, 3.1) 3.4)  8 2.41 ddq(12.0; 5.0; 2.56ddq(11.9; 4.4; 45.2 6.9) 7.0  9 202.8 10 7.02 d(15.4) 6.92 d(15.4) 126.711 6.20 dd(15.4; 9.4) 6.35 dd(15.4; 9.4) 144.1 12 3.43 dd(9.4; 2.0) 3.43dd(9.4; 1.9) 59.0 13 2.99 dd(9.3; 2.0) 3.06 dd(9.3; 1.9) 59.2 14 1.37 brm 1.42 br m 49.8 15 5.21 dq(10.8; 6.3) 5.40 dq(10.9; 6.2) 68.7 16 1.25d(6.3) 1.36 d(6.2) 17.4 17 1.15 d(6.8) 1.26 d(6.7) 18.4 18 0.89 d(7.5)1.02 d(6.8) 16.6 19 1.10 d(6.9) 1.18 d(7.0) 16.8 20 3.59 dd(10.3; 3.70dd(10.1; 67.1 2.8)/3.94 dd(10.3, 2.7/4.14 dd(10.1, 2.9) 2.9)  1′ 4.39d(8.0) 4.60 d(7.9) 103.0  2′ 3.39 dd(8.0; 6.2) 3.54 d(7.9) 70.8  3′ 1.32dd(12.3; 1.44 dd(11.8; 75.4 8.3)/1.38 dd(12.3; 8.3)/1.55 dd(1.8; 2.4)2.4)  4′ 36.3  5′ 3.73 ddq(8.3; 2.4; 3.90 ddq(8.3; 2.4; 66.6 6.2) 6.1) 6′ 3.70 dq(6.3; 3.9) 3.90 q(6.5) 68.5 OH-2′ 4.75 d(6.2) nd OH-3′ 3.89br s nd Me-5′ 1.07 d(6.2) 1.18 d(6.1) 20.4 OH-6′ 4.47 d(3.9) nd Me-6′0.98 d(6.3) 1.17 d(6.5) 15.7  1″ 4.49 d(8.1) 4.60 d(8.0) 101.2  2″ 3.02dd(8.1; 2.7) 3.11 dd(8.0; 2.9) 81.8  3″ 3.66 dd(2.7; 2.6) 3.80 dd(2.9;2.8) 80.5  4″ 3.09 ddd(9.6; 7.1; 3.20 ddd(9.5; 2.8) 73.6 2.6)  5″ 3.54dq(9.6; 6.2) 3.69 dq(9.5; 6.3) 70.1 OMe-2″ 3.41 s 3.57 s 58.5 OMe-3″3.46 s 3.60 s 61.1 OH-4″ 4.88 d(7.1) nd Me-5″ 1.12 d(6.2) 1.25 d(6.3)17.1nd: not detected

TABLE 12 Physicochemical Properties of Products P8 and P10a P8 P10aAppearance Colorless powder Colorless powder Chemical formula C₃₆H₅₈0₁₄C₃₇H₅₆0₁₅ Molecular weight 714 740 Nano-ESI-MS (m/z) Positive 737.4 [M +Na]⁺, 763.44 [M + Na]⁺, mode 563.3, 545.2, 589.27., 563, 389.2, 371.1,289.1 571.24, 559.19, 545.29, 417.16, 389.16, 371.16, 289.02 UVλ_(max)nm 216.73, shoulder 216.73, shoulder in MeOH 245.71 and 280 245.71 and278.09 IRγ_(max) diamond cell 3431, 2970, 2931, 3361, 2968, 2920, (cm⁻¹)2880, 1712, 1689, 2851, 1799, 1712, 1654, 1620, 1581, 1653, 1623, 1593,1454, 1416, 1382, 1562, 1508, 1457, 1354, 1326, 1279, 1417, 1383, 1356,1263, 1236, 1172, 1324, 1282, 1238, 1159, 1117, 1082 1194, 1173, 1159,1117, 1084, 1047 Relative solubility Highly MeOH, EtOR, Me₂CO, MeOH,EtOH, Me₂CO, Soluble n-BuOH, 1-PrOH, 2- n-BuOH, 1-PrOH, 2- PrOH, CH₂Cl₂,PrOH, CH₂Cl₂, EtOAc, CHCl₃ EtOAc, CHCl₃ Insoluble n-hexane, toluene,n-hexane, toluene, H₂O H₂O CCM (value of Rf)^(a) (I) 0.63 0.68 (II) 0.710.74 (III) 0.86 0.88 HPLC (Rt)^(b) 23.21 min 28.38 min^(a)CCM on silica gel (Merch No 5715). (I): EtOAc—MeOH (100:15). (II):n-BuOH—CH₃COOH—H₂O (3:1:1). (III): MeOH—CH₂Cl₂ (4:1).^(b)HPLC conditions: Uptisphere C₁₈ UP5ODB (250 × 7.8 mm, i.d.), Mobilephase: isocratic at 63% MeOH in H₂O, Flow rate: 1.5 ml/min, Detection:UV (220 nm).

TABLE 13 Assignments of NMR Data ¹H and ¹³C of the Product P10a(aldgamycin G) in CD₃OD at 298K P10a (aldgamycin G) Position δH (Multi,J in Hz) δC  1 166.3  2 6.03 d (15.5) 121.0  3 6.64 dd (15.5; 10.7)151.8  4 2.84 ddq (10.7; 10.2; 6.8) 41.8  5 3.42 d (10.2) 86.8  6 1.16 m34.5  7 1.56 m/1.82 ddd (14.0; 11.8, 2.9) 31.9  8 2.54 ddq (11.8; 4.8;7.0) 45.2  9 202.7 10 6.92 d (15.4) 126.6 11 6.35 dd (15.4; 9.4) 144.212 3.43 dd (9.4; 2.1) 59.0 13 3.06 dd (9.3; 2.1) 59.2 14 1.43 dm (10.8)49.8 15 5.40 dq (10.8; 6.3) 68.7 16 1.36 d (6.3) 17.4 17 1.24 d (6.8)18.2 18 1.01 d (6.8) 16.6 19 1.18 d (7.0) 16.8 20 3.70 dd (10.2,3.1)/4.13 dd (10.2; 2.29) 67.1  1′ 4.59 d (7.7) 102.4  2′ 3.47 d (7.7)70.6  3′ 86.6  4′ 1.61 dd (14.6; 11:1)/1.92 dd (14.6; 2.1) 41.1  5′ 3.84ddq (11.1; 6.1; 2.1) 67.0  6′ 4.50 q (6.5) 82.2  7′ 156.6 OH-2′ nd Me-5′1.23 d (6.1) 19.9 Me-6′ 1.57 d (6.5) 12.6  1″ 4.60 d (8.0) 101.2  2″3.11 dd (8.0; 2.8) 81.8  3″ 3.80 dd (2.8; 2.7) 80.5  4″ 3.20 dd (9.6;2.7) 73.6  5″ 3.69 dq (9.6; 6.2) 70.1 OMe-2″ 3.57 s 58.5 OMe-3″ 3.60 s61.1 OH-4″ nd Me-5″ 1.25 d (6.2) 17.1nd: not detected

TABLE 14 Assignments of NMR Data ¹H and ¹³C of the Product P10b(swalpamycine B) in CD₃OD at 298K P10b (swalpamycine B) Position δH(Multi, J in Hz) δC  1 166.7  2 5.88 d (15.4) 121.4  3 6.62 dd (15.4;10.0) 152.5  4 2.79 m 41.3  5 3.36 br d (10.1) 87.3  6 1.21 m 34.5  71.59 m^(a)/1.70 ddd (14.4, 11.7, 3.2) 31.9  8 2.53 m 45.3  9 205.7 106.46 d (15.0) 123.9 11 7.08 dd (15.0; 10.9) 142.4 12 6.28 dd (15.2;10.9) 133.4 13 6.08 dd (15.2; 9.3) 142.0 14 2.46 m 51.5 15 5.12 dq(10.1; 6.3) 69.7 16 1.39 d (6.8) 17.6 17 1.22 d (6.3) 19.1 18 1.01 d(6.8) 16.9 19 1.18 d (7.0) 17.0 20 3.66 dd (9.9; 3.6)/4.01 dd (9.9, 3.4)68.5  1′ 4.56 d (8.0) 102.9  2′ 3.54 d (8.0) 70.8  3′ 75.4  4′ 1.46m/1.54 m 36.3  5′ 3.87 ddq (8.3; 6.2; 2.4) 66.6  6′ 3.90 q (6.6) 68.5OH-2′ nd OH-3′ nd Me-5′ 1.19 d (6.2) 20.4 OH-6′ nd Me-6′ 1.16 d (6.6)15.6  1″ 4.61 d (8.0) 101.3  2″ 3.08 dd (8.0; 2.9) 81.8  3″ 3.77 dd(2.9; 2.8) 80.6  4″ 3.19 dd (9.6; 2.9) 73.6  5″ 3.67 dq (9.6; 6.3) 70.0OMe-2″ 3.53 s 58.6 OMe-3″ 3.59 s 61.1 OH-4″ nd Me-5″ 1.24 d (6.3) 17.1nd: not detected

1-25. (canceled)
 26. Actinomycete strain Saccharothrix SA registered atCNCM on 16 Feb. 2004 under number I-3160 or a mutant strain thereof. 27.Method for selecting the actinomycete strain Saccharothrix SA 103according to claim 26 and/or at least one of its mutant strains,characterized in that it comprises the following steps: a) contacting ofa biological sample likely to contain the said strain and/or at leastone of its mutant strains with an appropriate selection medium; b)isolation of the said strain and/or at least one of its mutant strains.28. Method for producing a culture medium from a culture of theactinomycete strain Saccharothrix SA 103 and/or at least one of itsmutant strains according to claim 26, characterized in that it comprisesthe following steps: a) fermentation of the said strain in a nutrientmedium to obtain the culture medium; b) optionally, separation of theculture medium obtained in step a).
 29. Method for producing a culturemedium according to claim 28, characterized in that the separationcarried out in optional step b) is a centrifugation and/or a filtrationand/or a pasteurization.
 30. Culture medium capable of being obtained bythe method according to claim
 28. 31. Method for producing an activeconcentrate from the culture medium according to claim 30, characterizedin that it comprises the following steps: a) organic extraction of theculture medium with an organic solvent; b) optionally, dehydration ofthe organic phase obtained and/or drying in vacuo; c) optionally,placing of the active concentrate in suspension, preferably filtrationof the suspension obtained, arid repetition of the steps a) and b) oforganic extraction and dehydration.
 32. Active concentrate capable ofbeing obtained by the method of claim
 31. 33. Method for producing anactive compound from the active concentrate according to claim 32 byreverse phase high performance liquid chromatography (reverse phaseHPLC), preferably preceded by thin layer chromatography and/or lowpressure liquid chromatography.
 34. Method for producing an activecompound according to claim 33, characterized in that the activecompound is a mutactimycin such as mutactimycin P11, mutactimycin PR,mutactimycin G or mutactimycin F, or an aldgamycin such as aldgamycin G,aldgamycin H or aldgamycin P10b, or the pharmaceutically acceptableaddition salts, isomers, enantionmers, diastereoisomers, and mixtures ofthese active compounds.
 35. Active compound capable of being obtained bythe production method according to claim 33, characterized in that theactive compound is mutactimycin PR having the following formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.
 36. Active compound capable ofbeing obtained by the production method according to claim 33,characterized in that the active compound is mutactimycin F having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.
 37. Active compound capable ofbeing obtained by the production method according to claim 33,characterized in that the active compound is mutactimycin G having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.
 38. Active compound capable ofbeing obtained by the production method according to claim 33,characterized in that the active compound is aldgamycin G having thefollowing stereochemical formula:

or its pharmaceutical acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.
 39. Active compound capable ofbeing obtained by the production method according to claim 33,characterized in that the active compound is aldgamycin H having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.
 40. Active compound according toclaim 39, characterized in that the aldgamycin H has the followingstereochemical formula:

or its pharmaceutical acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.
 41. Active compound capable ofbeing obtained by the production method according to claim 33,characterized in that the active compound is aldgamycin P10b having thefollowing formula:

or its pharmaceutically acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.
 42. Active compound according toclaim 41, characterized in that the aldgamycin P10b has the followingstereo chemical formula:

or its pharmaceutical acceptable addition salts, isomers, enantiomers,diastereoisomers, and mixtures thereof.
 43. Pharmaceutical compositioncontaining a therapeutically effective quantity of an active compoundaccording to claim 35 and a pharmaceutical acceptable excipient. 44.Active compound according to claim 35 for its use as a medicinalproduct.
 45. Method for the production of an antibiotic for preventingand/or treating an infection implicating a gram positive bacterium, suchas a streptococcus, a neonatal infection, a urinary infection, anendocarditis, a pneumonia, a meningitis, an otitis, a listeriosis,diphtheria, tuberculosis or leprosy, comprising an effective amount of apharmaceutical composition according to claim
 43. 46. Method for theproduction of an antiviral medicinal product preventing and/or treatingan infection implicating the acquired immune deficiency syndrome (AIDS)virus, the vaccine virus, corona virus, papillomavirus, parvovirus,virus of catarrhal fever of sheep, dengue virus, Ebola virus, orinfluenza, smallpox, measles, rubella, varicella, hepatitis A, B, C, Dor E, mononucleosis, yellow fever, encephalitis or herpes, comprising aneffective amount of a pharmaceutical composition according to claim 43.47. Method for the production of an anticancer medicinal product forpreventing and/or treating a subject suffering from a cancer, such ascancer of the lung, the uterus, breast or ovary, colorectal cancer,leukemia or a subject suffering from a tumour of the prostate, thebladder, skin, brain, throat, comprising an effective amount of apharmaceutical composition according to claim
 43. 48. Method forpreventing or treating a disease in a plant using a phytopharmaceuticalproduct comprising an active compound according to claim 35.